1. Field of the Invention
The present invention relates generally to the diagnosis, management and therapy of central nervous system disorders such as stroke, transient ishemic attack, and traumatic brain injury. In particular, the invention relates to methods and kits for evaluating these central nervous system disorders, in order to better respond to episodes of focal cerebral ishemia, and to best manage the risk associated with future acute incidences.
2. Background Information
Stroke or “brain attack” is clinically defined as a rapidly developing syndrome of vascular origin that manifests itself in focal loss of cerebral function. In more severe situations, the loss of cerebral function is global. A stroke occurs when the blood supply to the part of the brain is suddenly interrupted (ischemic) or when a blood vessel in the brain bursts, spilling blood into the spaces surrounding the brain cells (hemorrhagic). The symptoms of stroke are easy to spot: sudden numbness or weakness, especially on one side of the body; sudden confusion or trouble speaking or understanding speech; sudden trouble seeing in one or both eyes; sudden trouble walking; dizziness; or loss of balance or coordination. (National Institute of Neurological Disorders and Stroke, 2001). Stroke is the most common devastating neurologic disease in the world, and the third leading cause of death in world after heart disease and cancer. Despite recent progress understanding stroke mechanisms, stroke management is still not optimal for a number of reasons.
The importance of promptly diagnosing a stroke after symptoms appear cannot be overstated. Delays in diagnosis and medical intervention beyond 3 hours after stroke onset may contribute to clinical deterioration and disability. An early diagnosis enables doctors to more effectively choose the emergency intervention such as anti-platelet or/and neuroprotective therapy, and also to make better prognoses of disease outcome. Successful treatment of stroke requires rapid state diagnosis. The delay in achieving an accurate and certain diagnosis wastes the limited amount of time available in which the brain can respond to reperfusion, and significantly increases the risk of hemmorrhage after most of the permanent injury has occurred (Marler J. R. Annl. Emergency Med. 1999, 33: 450-451).
Unfortunately, however, many people who have a stroke either do not seek immediate medical care or suffer from delays in medical care even in countries where stroke care is advanced, such as the United States and Europe (Alberts M J, Hademenos G, Latchaw R E, et al. JAMA 2000; 23:3102-3109). Several clinical criteria can be employed to diagnose whether a patient is having a stroke, but even all these criteria do not always allow one to differentiate the episode from other disorders, such as epilepsy, syncope, and migraine (Toole J F. Cerebrovascular Disorders. 1999. Lippincott, Williams & Wilkins, New York, 5th Ed., 542 p). Moreover, progressing stroke is only partially predictable based on clinical and neuroimaging data that is currently available to neurologists.
Transient ischemic attack (TIA) is a short-lived episode of focal neurologic deficit which often precedes the cerebral infarction of a stroke. It occurs when the blood supply to part of the brain is briefly interrupted, and is typically accompanied by permanent brain damage (albeit less severe damage than normally results from a stroke). TIA symptoms, which usually occur suddenly, are similar to those of stroke but do not last as long. Most symptoms of a TIA disappear within an hour, although they may persist for up to 24 hours. Symptoms can include: numbness or weakness in the face, arm, or leg, especially on one side of the body; confusion or difficulty in talking or understanding speech; trouble seeing in one or both eyes; and difficulty with walking, dizziness, or loss of balance and coordination. (National Institute of Neurological Disorders and Stroke, 2001). Patients who have suffered a TIA have 9.5 times greater risk of having a future stroke than those who have not had a TIA, and about one third of patients who suffer a TIA will have an acute stroke in the future. (American Stroke Association, 2001). However, because the symptoms of TIA are short term, many patients do not recognize the event as a TIA or perceive the event as a warning of a potentially impending stroke.
Standard treatments to reduce the risk of future stroke include the use of antiplatelet agents, particularly aspirin. People with atrial fibrillation (irregular beating of the heart) may be prescribed anticoagulants. The most important treatable factors linked to TIAs and stroke are high blood pressure, cigarette smoking, heart disease, carotid artery disease, diabetes, and heavy use of alcohol. Lifestyle changes can often be implemented to reduce these factors. However, it is necessary to diagnose the TIA as a warning sign of impending stroke before such treatments can be administered. Therefore, a laboratory blood test to detect TIA or stroke, or the risk of suffering a TIA or stroke in the future, would be of tremendous benefit.
During the past 5 years a number of molecular and immunochemical assays have been evaluated for clinical use in neurology. (Schenone A. et. al. Current Opinion in Neurology. 1999, 12: 603-604; Honnorat J. J. Neurol. Neurosurg. Psychiatry. 1996, 61:270-278). At present, the Thrombogene V and two Thrombx tests are available for diagnosing stroke/thrombosis from Athena Diagnostic. These tests evaluate the frequent deep vein thrombosis and hypercoagulation states of patients to evaluate the need for intravenous anticoagulant therapy. The Thrombogene V test detects the Factor V Leiden mutation by Polymerase Chain Reaction (PCR) in the blood of patients. The other two tests monitor changes of different blood coagulation markers: antithrombin m protein C, factor IX, and anticardiolipin antibodies (IgG, IgM, IgA) by use of ELISA technique. These tests thus reveal the hypercoagulation state as a result of a thrombotic events, such as stroke stroke.
Stroke can be related to different types of venous thromboembolisms, which are common disorders with considerable morbidity and potential for mortality (Anderson, D.; Wells, P. Cur. Opinion in Hemat. 2000, 7: 296-301). The biochemical marker: D-dimer, a breakdown product of a cross-linked fibrin blood clot that indicates the occurrence of plasmin mediated lysis of cross-linked fibrin, has been extensively evaluated for use in diagnostic tests for indicating acute venous thromboembolism. Indeed, a fully automated, semi-quantative latex agglutination assays that uses turbimetric or agglutination endpoints has been developed that provides results within 20 minutes with sensitivity between 89% and 95% (Roussi J.; Bentolila L.; Contribution of D-dimer determination in the exclusion of deep venus thrombosis in spinal cord injury patients. Spinal Cord, 1999; v.37: p.548-552). Unfortunately, however, the presence of D-dimer may also be increased in other settings that result in fibrin generation, including recent surgery, hemmorhage, trauma, cancer, and pregnancy (Anderson D R., Wells P S.; Thromb. Haemost.; 1999; 82:878-886).
However, the foregoing tests do not elucidate upon the TIA/stroke mechanisms that are actually responsible for the damage associated with neurotoxic molecular events. It is necessary to find out specific and sensitive biomarkers which could be helpful to recognize initial brain damage and which could help to choose not only the appropriate anticoagulant treatment, but also emergency or regular neuroprotective therapy.
It is well known that two of the three leading causes of death, namely cardiovascular diaseses and stroke, are the end result of atherosclerosis. Thus, it is not surprising that several biochemical markers implicated in thromboembolic processes are also reported to be associated with stroke and stroke risk. Among these are homocysteine, cholesterol and LDL (Cerebrovascular Disorders ed. by J. E. Toole. Lippincott Williams & Wilkins. 1999, pp.34-35), which are also classified as risk factors to cardiovascular and cerebrovascular diseases. (Hankey G J., and Eikelboom J W. Lancet. 354: 407-413 (1999). Approximately one fourth of patients with symptomatic atherosclerosis have elevated plasma homocysteine levels caused by various factors. High levels of homocysteine may run in families with increased susceptibility to heart attack and stroke (Graham I. J. Ir. Call. Phys. Surg. 1995; 24: 25-30). Elevated plasma homocysteine may be a causal and modifiable risk-factor for ischemic stroke, but the results of previous studies have been conflicting (Deulofeu V N R, Chamorro A, Piera C. Med Clin (Barc). 1998; 110: 605-608; Yamamoto T, Rossi S, Stiefel M E, Doppenberg E, Zauner A, Bullock R, Marmarou A. Acta Neurochir. Suppl. (Wien) 1999; 75:17-19).
The neurotoxic effect of excitatory amino acids (glutamate, aspartate) in the brain has also been well documented. The results of this work show a correlation between glutamate content in the blood and the severity of acute ischaemia (Castillo J, Dávalos A, Naveiro J, Noya M. Stroke 1996, 27:1060-1065; Castillo J, Dávalos A, Noya M. Lancet. 1997; 349:79-83). Cerebral damage and its association with progressing stroke has been attributed to increased glutamate release, or low glutamate reuptake, both in animals and in humans (Dávalos A, Castillo J. Serena J. Noya M. Stroke 1997; 28:708-710).
However, only 56% of patients with progressing stroke are reported to have high glutamate content in their blood serum (Dávalos A., Toni D., Iweins F., et al., 1999, 30: 2631-2636). Moreover, even though glutamate is considered the strongest biochemical predictor of progressing stroke (Dávalos A, and Castillo J. In Book: Cerebrovascular Disease. Current Med. Inc.: Philadelphia. 2000 Chapter 16, pp.169-181), this marker remains non-specific for TIA. Glutamate changes have also been observed in the blood of patients with epilepsy and other nervous system disorders (Meldrum B S. J. Nutrition. 2000, 130:1007S-1015S).
Over the last three decades substantial progress has been made in elucidating the mechanisms by which cerebral ischemia leads to brain damage. The cellular and molecular mechanisms of cerebral ischemia abnormalities have been better defined through the role of glutamate and glutamate receptors, one of the most distributed excitatory neuroreceptors in brain, in regulating of initial stages of brain damage. Indeed, numerous molecular investigators consider glutamate receptors to be one of the key biological receptors involved in the molecular mechanisms of TIA and stroke (Meldrum B S. J. Nutrition. 2000, 130:1007S-1015S). According to a leading hypothesis, ischemia-induced glutamate release activates these glutamate receptors. It has been shown that glutamate and homocysteine (the sulfinic analog of aspartate) activate the glutamate binding site of NMDA receptors and participate in neurotoxic processes (Lipton S. A., Kim W. K., Choi Y. B., Kumar S., et al. PNAS. 1997, 94: 5923-5928).
Glutamate receptors are divided into two main groups: ionotropic and metabotropic. The ionotropic neuroreceptors are ligand-gated ion channels that are subdivided into NMDA, AMPA and kainate receptor subtypes. There are four NR2 subunits: NR2A, NR2B, NR2C and NR2D, which is responsible for Ca2+-permeability regulation. NMDA receptors can be modified by ischemia, resulting in changes of ion permeability and/or ion selectivity.
Recent research findings indicate that the blood of patients with CNS disorders other than TIA or stroke exhibit properties of autoimmunization to products of nerve cell degradation (Vincent A., Oliver L., Pallace J. J Neuroimmun. 1999; 100: 169-180). For example, a correlation between AMPA GluR1 autoantibodies and common epilepsy has been shown (Dambinova et al. J. Neurol. Sci. 1997; 152: 93-97; Dambinova et al. J. Neurochem. 1998;71: 2088-2093), as has a correlation between AMPA GluR 3 autoantibodies and Rasmussen's encephalitis (Rogers S W, Andrews P I, Gahring L C, et al. Science. 1995;265:648-651; Twyman R E, Gahring L C, Spiess J, Rogers S W. Neuron. 1995; 14:755-762; Gahring L C, Twyman R I, Greenlee J E, Rogers S W. Mol. Med. 1995; 1:245-253).
In a similar vein, several researchers have reported an increase in NMDA receptor synthesis, the appearance of high levels of receptor antigen, and the generation of autoantibodies to the receptors during the initial stages of cerebral ishemia (Gusev et al., J. Neurol.& Psych. 1996, 5:68-72; Dambinova et al. J. Neurol. Sci. 1997, 152:93-97; Dambinova et al. J. Neurochem. 1998, 71: 2088-2093). Acting on this research, one company developed a laboratory kit (cerebral ischaemia (CIS)-test) that detects autoantibodies to the N-terminus domain of the NR2A subunit in the blood of patients with TIA or stroke (Gusev E. L., Skvortsova V I, Alekseev A A, Izykenova G A, Dambinova S A. S. S Korsakov's J. Neurol.& Psych. 1996; 5:68-72). The N-terminus domain of the NR2A subunit of NMDA receptors was selected as the immunoreactive epitope on the basis of molecular biological and experimental studies showing that this epitope is the most immunoreactive region of the receptor (Dambinova S A, Izykenova G A. J. High Nervous Activity. 1997; 47:439-446).
More recently, researchers have reported a correlation between the effectiveness of a stroke treatment regimen and the levels of autoantibodies to the NR2A and NR2B subunits of NMDA. In particular, these researchers have reported increased titers of autoantibodies to the NR2A and NR2B subunits of NMDA in the blood of patients severely affected by stroke, and a reduction of the autoantibodies, accompanied by an improvement in neurological function, during therapy by glycine—a non-specific agonist of NMDA receptors (Gusev et. al. Cerebrovascular Diseases. 2000, 10: 49-60). Patients that responded positively to glycine had lower autoantibody titers than patients who were not treated, and had levels of autoantibodies that were close to the levels of autoantibodies in control subjects.
Unfortunately, the use of NR2A and NR2B autoantibodies in the diagnosis of stroke or TIA does not provide a real-time assessment of the damage being done by a stroke or TIA. Rather, because of the time the immune system requires to mount an immune response, and to generate NR2A and NR2B autoantibodies, methods that test for these antibodies at best provide a delayed assessment of the extent and severity of stroke or TIA.
Investigators from Canada (Hill M. D., Jackowski G., Bayer N., Lawrence M., Jaeschke R. Can. Med. Assoc. J. 2000, 163: 1139-1140) have proposed a new diagnostic laboratory assay for differentiating stroke subtype. They designed a preliminary prospective cohort study to test a panel of biochemical markers (neuron-specific enolase[NSE], myelin basic protein [MBP], S-100 [betta] protein and thrombomodulin [Tm]) in blood samples from patients with acute ischemic stroke. These markers were chosen because they cover important cellular components of the brain that might be damaged in acute stroke. The 4 biochemical markers were assayed using a standard ELISA technique.
The results of this investigation demonstrated elevated levels of NSE in 89% of the patients admitted in hospitals, Tm in 43%, MBP in 39% and S-100 [beta] in 32%. At least one of the markers was elevated on admission in 93% of the acute stroke patients. By stroke type, 100% of the patients with lacunar stroke, 100% of those with posterior circulation stroke and 90% of those with partial anterior circulation stroke had elevated NSE levels on admission. Conversely, none of the patients with lacunar stroke had en elevated S-100[beta] level initially or subsequently. Peak levels of NSE, S-100 [beta] and MBP, but not of Tm, were significantly correlated with admission NIHSS scores (p<0.05).
For stroke, 3 hours is an outside limit for administering appropriate therapies. The focus must change from extensive evaluation before any action to a well-planned acute emergency therapy developed using an appropriate diagnostic strategy. Every future advance to improve the outcome after TIA/stroke will depend on a fast initial response-within minutes and not hours (Marler J. R. Annl. Emergency Med. 1999, 33: 450-451). Therefore, it is especially important to develop a fast and simple method (within one hour) of detecting brain and blood biomarkers capable of recognizing the initial processes of TIA/stroke before irreparable ischemic damage ensues.